JP2023109906A - PROTEIN BINDERS FOR iRHOM2 - Google Patents

Abstract

To provide a protein binder that binds to human iRhom2.SOLUTION: The present invention relates to a protein binder that binds to human iRhom2, and inhibits and/or reduces TACE/ADAM17 activity when bound to human iRhom2. According to one embodiment, the protein binder is a monoclonal antibody, or a target-binding fragment or derivative thereof retaining target binding capacities, or an antibody mimetic.SELECTED DRAWING: Figure 2

Description

This application relates to protein binders for iRhom2.

ADAM metallopeptidase domain 17 (ADAM17) (NCBI reference for human ADAM17: NP_003174), also called TACE (tumor necrosis factor-alpha converting enzyme), is a 70 kDa enzyme belonging to the ADAM protein family of disintegrins and metalloproteases. It is a polypeptide of 824 amino acids.

ADAM17 is understood to be involved in the processing of tumor necrosis factor-α (TNF-α) on the cell surface and from within the intracellular membrane of the trans-Golgi network. This process, also known as "shedding", involves cleavage and release of soluble ectodomains from membrane-bound proproteins (such as proTNF-α) and is physiologically important. It is known that ADAM17 was the first "sheddase" identified and is also understood to play a role in the release of diverse membrane-anchored cytokines, cell adhesion molecules, receptors, ligands, and enzymes.

Cloning of the TNF-α gene revealed that it encodes a 26 kDa type II transmembrane propolypeptide that becomes inserted into the plasma membrane upon translocation within the endoplasmic reticulum. At the cell surface, pro-TNF-α is biologically active and can induce immune responses through contact secretory intercellular signaling. However, proTNF-α can undergo proteolytic cleavage at its Ala76-Val77 amide bond, which releases a soluble 17 kDa extracellular domain (ectodomain) from the proTNF-α molecule. This soluble ectodomain is a cytokine commonly known as TNF-α and is crucial in the paracrine signaling of this molecule. This proteolytic release of soluble TNF-α is catalyzed by ADAM17.

ADAM17 also regulates the MAP kinase signaling pathway by regulating cleavage of the EGFR ligand amphiregulin in the mammary gland. In addition, ADAM17 is involved in shedding of the cell adhesion molecule L-selectin.

Recently, ADAM17 was discovered as a critical mediator of resistance formation to radiotherapy. Radiation therapy can induce a dose-dependent increase in furin-mediated cleavage of the ADAM17 proform to active ADAM17, resulting in enhanced ADAM17 activity in vitro and in vivo. In non-small cell lung cancer, radiation therapy activates ADAM17, which results in shedding of multiple survival factors, activation of growth factor pathways, and resistance to radiation therapy.

ADAM17 has become of interest as a therapeutic target molecule as it appears to be a crucial factor for the release of different pathogenic and non-pathogenic factors, including TNFα. Therefore, various attempts have been made to develop ADAM17 inhibitors.

However, to date, no such inhibitor has proven clinically successful.

It is therefore an object of the present invention to provide new approaches that allow regulation, modulation, reduction or inhibition of ADAM17 activity.

It is another object of the present invention to provide new approaches that enable the treatment of inflammatory diseases.

These and other objects are solved by the features of independent claims. The dependent claims disclose embodiments of the invention which may be preferred in certain circumstances. Likewise, the specification discloses further embodiments of the invention that may be preferred in certain circumstances.

The present invention provides, inter alia, protein binders that bind to human iRhom2, wherein binding to human iRhom2 inhibits and/or reduces TACE/ADAM17 activity.

FIG. 1 shows the sequences of peptides used herein for immunization and peptide binding ELISA analysis. These peptides are subsequences of the entire iRhom2 or iRhom1 sequence. To enhance immunogenicity, some peptides have been conjugated to KLH (keyhole limpet hemocyanin) via the SH group of cysteine. For peptide binding analysis, these peptides are alternatively conjugated to biotin. To that end, either the naturally occurring cysteine was used at either the N- or C-terminus of each peptide, or a cysteine was added to either the N- or C-terminus (denoted "-C-" in Figure 1). ). To avoid non-specific formation of intrachain disulfide bonds or intrachain binding of KLH and/or biotin, intrachain cysteines were replaced with aminobutyric acid (labeled "Abu" in Figure 1).

FIG. 2 shows the results of a TNFα release assay (shedding assay) for functional screening of hybridoma supernatants, demonstrating that hybridoma clone 4H8 supernatant effectively inhibited LPS-induced TNFα shedding in THP-1 cells. Prove to interfere.

Figure 3 shows the results of an ELISA analysis for antibody isotyping showing that the antibody 4H8-E3 of the invention is of the mouse IgM isotype.

FIG. 4 shows the antibody 4H8-E3 of the invention recognizing an epitope within a portion of the large extracellular loop 1 of human iRhom2 (the “juxtamembrane domain”, JMD) adjacent to the first “transmembrane domain” (TMD1). Results of peptide binding ELISA assays revealing .

Antibody 4H8-E3 of the invention recognizes peptide 3, which corresponds to amino acids 431 to 459 of human iRhom2, which is the JMD portion ("juxtamembrane domain") of the large extracellular loop 1 of human iRhom2 that flanks TMD1.

FIG. 5 is a peptide binding ELISA demonstrating that the antibody 4H8-E3 of the invention recognizes an epitope within the extracellular juxtamembrane region adjacent to TMD1 of human iRhom2, but not within the homologous region of human iRhom1. Shows the results of the analysis. Antibody 4H8-E3 of the invention recognizes peptide 3, but not peptide 3b, which corresponds to the respective homologous portion of human iRhom1.

Figure 6 shows the results of a TNFα release assay demonstrating that antibody 4H8-E3 of the invention inhibits LPS-induced shedding of TNFα in THP-1 cells.

FIG. 7 shows the results of a TNFα release assay demonstrating concentration-dependent inhibition of LPS-induced TNFα shedding by the antibody 4H8-E3 of the invention in THP-1 cells.

FIG. 8 is a schematic diagram of iRhom2 illustrating the location of the juxtamembrane domains adjacent to TMD1 (A), Loop1 (B) and the C-terminus (C).

Figure 9 shows the amino acid sequence of human iRhom2 as set forth in SEQ ID NO: 16, along with sequences corresponding to immunizing peptides used in the present invention.

FIG. 10 shows an alignment of human iRhom2 as set forth in SEQ ID NO:16 and human iRhom1 as set forth in SEQ ID NO:17. The gray area indicates the sequence corresponding to immunizing peptide 3 used in the present invention.

(detailed description)
According to one aspect of the invention there is provided a protein binder that binds to human iRhom2, wherein binding to human iRhom2 inhibits and/or reduces TACE/ADAM17 activity.

Rhomboid family member 2 (iRhom2) is a protein encoded by the RHBDF2 gene in humans. It is a transmembrane protein consisting of about 850 amino acids and has 7 transmembrane domains. The inventors of the present invention have demonstrated for the first time that iRhom2 serves as a target for protein binders to inhibit TACE/ADAM17 activity.

iRhom2 has different isoforms. The experiments done here were established using the isoform defined as NCBI reference NP_078875.4. However, the teachings are transferable to other isoforms of iRhom2, as shown, but not limited to, in the table below:

As used herein, the term "inhibits and/or reduces TACE/ADAM17 activity" refers to protein binders that block or reduce the activity of TACE/ADAM17, e.g., as measured in the respective shedding assay. (see, eg, FIG. 2 and Example 5).

According to one or more embodiments, the protein binder is a monoclonal antibody, or a target-binding fragment or derivative thereof that retains target-binding ability, or an antibody mimetic.

As used herein, the term "monoclonal antibody (mAb)" refers to an antibody composition having a homogenous population of antibodies, i.e., a homogenous population of immunoglobulins or fragments or derivatives thereof that retain target-binding ability. . Particularly preferred such antibodies are selected from the group consisting of IgG, IgD, IgE, IgA and/or IgM, or fragments or derivatives thereof that retain target binding ability.

As used herein, the term "fragment" refers to fragments of such antibodies that retain target-binding ability. for example,
● CDR (Complementarity Determining Region)
● hypervariable regions,
● Variable domain (Fv)
- IgG or IgM heavy chain (consisting of VH, CH1, hinge, CH2 and CH3 regions)
- IgG or IgM light chain (consisting of VL and CL regions), and/or - Fab and/or F(ab) ₂ .

The term "derivative" as used herein refers to protein constructs that are structurally different but still have some structural relatedness, such as scFv, Fab and/or F(ab) ₂ , as well as double , triple or higher specificity antibody constructs, and also refer to common antibody concepts that retain target binding ability. All of these items are described below.

Other antibody derivatives known to those skilled in the art are diabodies, camelid antibodies, nanobodies, domain antibodies, bivalent homodimers with two chains composed of scFvs, IgAs (linked by a J chain and a secretory component). two IgG structures), shark antibodies, antibodies consisting of New World primate frameworks and non-New World primate CDRs, dimerization constructs comprising CH3+VL+VH, and antibody conjugates (e.g., toxins, cytokines, radioisotopes or antibody or fragment or derivative) linked to a label. These types are well documented in the literature and can be used by those skilled in the art based on the present disclosure without further inventive effort.

Methods for producing hybridoma cells are disclosed in Koehler & Milstein (1975).

Methods for the production and/or selection of chimeric or humanized mAbs are known in the art. For example, US6331415 by Genentech describes the production of chimeric antibodies, while US6548640 by the Medical Research Council describes CDR grafting techniques and US5859205 by Celltech describes the production of humanized antibodies.

Methods for the production and/or selection of fully human mAbs are known in the art. These include the use of transgenic animals immunized with the respective protein or peptide, or the use of suitable display technologies such as yeast display, phage display, B-cell display or ribosome display, wherein libraries are screened against human iRhom2 in stationary phase.

In vitro antibody libraries are disclosed inter alia in US6300064 by MorphoSys and US6248516 by MRC/Scripps/Stratagene. Phage display technology is disclosed, for example, by Dyax in US5223409. Transgenic mammalian platforms are described, for example, in EP1480515A2 by TaconicArtemis.

IgG, IgM, scFv, Fab and/or F(ab) ₂ are antibody formats well known to those skilled in the art. Relevant enabling techniques are available from the respective textbooks.

As used herein, the term "Fab" relates to an IgG/IgM fragment containing the antigen-binding region, said fragment comprising one constant and one antibody from each heavy and light chain of the antibody. consists of one variable domain

As used herein, the term "F(ab) ₂ " refers to an IgG/IgM fragment consisting of two Fab fragments linked together by disulfide bonds.

As used herein, the term "scFv" is a single-chain variable region fragment that is a fusion of immunoglobulin heavy and light chain variable regions with a short linker, usually serine (S) or glycine ( G). This chimeric molecule retains the specificity of the original immunoglobulin despite removing the constant region and introducing a linker peptide.

Modified antibody formats are, for example, bi- or tri-specific antibody constructs, antibody-based fusion proteins, immunoconjugates, and the like. These types are well documented in the literature and can be used by one skilled in the art based on this disclosure and add further activity of the invention.

As used herein, the term "antibody mimetic" relates to organic molecules, most often proteins, that bind specifically to a target protein, like antibodies, but are not structurally related to antibodies. Antibody mimetics are usually artificial peptides or proteins with molar masses of about 3-20 kDa. Definitions include inter alia affibody molecules, affilins, affimas, affitins, alphabodies, anticalins, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, and nanoCLAMPs.

In one or more embodiments, the protein binder is an isolated antibody, or target-binding fragment or derivative thereof that retains target-binding ability, or an isolated antibody mimetic.

In one or more embodiments, the antibody is a modified or recombinant antibody, or a target-binding fragment or derivative thereof that retains target-binding ability, or a modified or recombinant antibody mimetic.

According to one or more embodiments of the present invention, inhibition or reduction of TACE/ADAM17 activity is caused by interference with iRhom2-mediated TACE/ADAM17 activation.

According to one or more embodiments of the invention, the antibody inhibits or reduces TNFα shedding.

As used herein, "TNFα shedding" refers to the process by which membrane-anchored tumor necrosis factor alpha (mTNFα/proTNFα) is released into the environment to become soluble TNFα (sTNFα or simply TNFα). . This process is specifically triggered by TACE/ADAM17.

According to one or more embodiments of the present invention, the human iRhom2 bound protein binders include:
a) an amino acid sequence set forth in SEQ ID NO: 16, or b) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 16, provided that it maintains iRhom2 activity.

In some embodiments, human iRhom2 is ≧81%, preferably ≧82%, more preferably ≧83%, ≧84%, ≧85%, ≧86%, ≧87%, ≧88 with SEQ ID NO:16 %, ≧89%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98 or most preferably ≧99% sequence identity contains amino acid sequences with specific properties.

SEQ ID NO: 16 represents the amino acid sequence of inactive rhomboid protein 2 (iRhom2) isoform 1 [Homo sapiens] accessible under NCBI reference NP_078875.4. In general, iRhom2 exists in different variants and isoforms. Similarly, there are or may be variants containing conservative or silent amino acid substitutions that maintain full or at least substantial iRhom2 activity. These isoforms, variants and mutants are encompassed within the identity ranges defined above, but exclude dysfunctional, inactive variants and mutants.

In this context, "conservative amino acid substitutions" have less effect on antibody function than non-conservative substitutions. Although there are many ways to classify amino acids, they are often classified into six major groups based on their structure and the general chemical properties of the R groups.

In some embodiments, a "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with the following side chains:
● basic side chains (e.g. lysine, arginine, histidine),
● acidic side chains (aspartic acid, glutamic acid, etc.),
● uncharged polar side chains (glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, etc.),
● Nonpolar side chains (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, etc.),
● β-branched side chains (threonine, valine, isoleucine, etc.)
● Aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine)

Other conservative amino acid substitutions can also occur across amino acid side chain families, such as replacing asparagine with aspartic acid to modify the charge of a peptide. Conservative changes can also include substitution of chemically homologous non-natural amino acids (ie, synthetic non-natural hydrophobic amino acids for leucine, synthetic non-natural aromatic amino acids for tryptophan).

A "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison window is the fraction of the optimal alignment of the two sequences. To that end, it may contain additions or deletions (ie, gaps) compared to a reference sequence (eg, polypeptide) that does not contain additions or deletions. The percentage is obtained by determining the number of positions where identical nucleobases or amino acid residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100. It is calculated by obtaining the percentage of sequence identity from the

The terms "identical" or "identity", in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are identical sequences. Two sequences, when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection: A specified percentage of identical amino acid residues or nucleotides (i.e., at least 85%, 90%, 95%, 96%, 97%, over a specified region, or, if not specified, over the entire sequence of the reference sequence) Two sequences are "substantially identical" if they have 98% or 99% sequence identity).

The disclosure provides polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein. Optionally, identity is over a region that is at least about 15, 25 or 50 nucleotides in length, or more preferably over a region that is 100-500 or 1000 or more nucleotides in length, or over the entire length of the reference sequence. exist for a long time.

With respect to amino acid sequences, identity or substantial identity may be at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length; Some can be at least about 150, 200 or 250 amino acids in length, or can span regions spanning the entire length of the reference sequence. For shorter amino acid sequences, e.g., amino acid sequences of 20 amino acids or less, substantial identity is achieved when 1 or 2 amino acid residues are conservatively substituted according to the conservative substitutions defined herein. exist.

According to one or more embodiments of the present invention, the protein binder binds to the extracellular juxtamembrane domain adjacent to transmembrane domain 1 (TMD1) of human iRhom2.

The juxtamembrane domain adjacent to transmembrane domain 1 (TMD1) is a region encompassing a stretch of amino acids C-terminal to the first transmembrane domain (TMD1). See FIGS. 8 and 9 for explanatory diagrams.

In one embodiment, the juxtamembrane domain flanking transmembrane domain 1 (TMD1) is amino acids 431-459 of the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 16 including.

In another embodiment, the juxtamembrane domain flanking transmembrane domain 1 (TMD1) is from amino acid 431 of the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 16. 447 included.

According to one embodiment of the present invention, the protein binder binds to an amino acid sequence of human iRhom2 comprising a) at least the amino acid sequence shown in SEQ ID NO:3 b) an amino acid having 90% or more sequence identity with SEQ ID NO:3 arrangement.

In some embodiments, SEQ ID NO:3 and ≧91%, preferably ≧92%, more preferably ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98 or most preferably Amino acid sequences with ≧99% sequence identity.

In one embodiment, the antibody binds the entire amino acid sequence as described above. In another embodiment, the antibody also binds to additional amino acid sequences of human iRhom2 outside of SEQ ID NO:3 or outside of amino acid sequences having at least 90% sequence identity with SEQ ID NO:3.

Further, depending on where the amino acid is located, the epitope bound by the antibody is either linear or conformational.

According to one or more embodiments of the invention, the protein binder binds to one or more amino acid sequences of human iRhom2 each comprising one or more amino acids within the amino acid sequence set forth in SEQ ID NO:3.

In one embodiment, the antibody binds to one distinct subsequence within SEQ ID NO:3 comprising one or more amino acids.

In one embodiment, the antibody binds to two or more distinct subsequences within SEQ ID NO:3, each of these subsequences comprising one or more amino acids

According to one or more embodiments of the present invention, the protein binder is A431, Q432, H433, V434, T435, T436, Q437, L438, V439, L440, R441, N442, K443, G444, V445, Y446, E447, S448, V449, K450, Y451, I452, Q453, Q454, E455, N456, F457, W458, V459, wherein the amino acid residue numbering is , to the amino acid sequence set forth in SEQ ID NO: 16 (human iRhom2).

In one or more embodiments, the protein binder is ≧2, ≧3, ≧4, ≧5, ≧6, ≧7, ≧8, ≧9, ≧10, ≧11, ≧12, ≧13, ≧14, ≧15, ≧16, ≧17, ≧18, ≧19, ≧20, ≧21, ≧22, ≧23, ≧24, ≧25, ≧26, ≧27, ≧28, or ≧ Binds to 29 amino acid residues. Each amino acid residue may occur in a separate contiguous sequence or in two or more clusters within SEQ ID NO:3.

In another embodiment, the protein binder binds to the amino acid sequence of human iRhom2 comprising at least the amino acid sequence set forth in SEQ ID NO:4 or having at least 90% sequence identity with SEQ ID NO:4. . The same fallback positions for sequence identity apply.

In one embodiment, the antibody binds the entire amino acid sequence as described above. In another embodiment, the antibody also binds to additional amino acid sequences of human iRhom2 outside of SEQ ID NO:4 or outside of amino acid sequences having at least 90% sequence identity with SEQ ID NO:4.

Further, depending on where the amino acid is located, the epitope bound by the antibody is either linear or conformational.

According to one or more embodiments of the present invention, the protein binder binds to one or more amino acid sequences of human iRhom2 each comprising one or more amino acids within the amino acid sequence set forth in SEQ ID NO:4

In one embodiment, the antibody binds to one distinct subsequence within SEQ ID NO:4 comprising one or more amino acids.

In one embodiment, the antibody binds to two or more distinct subsequences within SEQ ID NO:4, each of these subsequences comprising one or more amino acids.

According to one or more embodiments of the present invention, the protein binders are , K443, G444, V445, Y446, E447, S448, V449, K450, Y451, I452, Q453, Q454, E455, N456, F457, W458, V459. , where the amino acid residue numbering is relative to the amino acid sequence set forth in SEQ ID NO: 16 (human iRhom2).

In one or more embodiments, the protein binder is ≧2, ≧3, ≧4, ≧5, ≧6, ≧7, ≧8, ≧9, ≧10, ≧11, ≧12, ≧13, ≧14, ≧15, ≧16, ≧17, ≧18, ≧19, ≧20, ≧21, ≧22, ≧23, ≧24, ≧25, ≧26, ≧27, ≧28, ≧29 , ≧30, ≧31, ≧32, ≧33 or ≧34 amino acid residues. Each amino acid residue may occur in a separate contiguous sequence or in two or more clusters within SEQ ID NO:3.

According to one or more embodiments of the present invention, the protein binder is not cross-reactive with human iRhom1 or its juxtamembrane domain adjacent to transmembrane domain 1 (TMD1).

According to one or more embodiments of the present invention, the protein binder is cross-reactive with mouse iRhom2, or its juxtamembrane domain adjacent to transmembrane domain 1 (TMD1).

According to one or more embodiments of the present invention, the protein binder is an antibody in at least one format selected from the group consisting of IgG, scFv, Fab, (Fab)2.

According to one or more embodiments of the invention, the protein binder is an antibody with an isotype selected from the group consisting of IgG, IgM.

According to one or more embodiments of the present invention, protein binders are murine, chimerized, humanized, or human antibodies.

According to one embodiment of the invention the protein binder is the antibody 4H8-E3. In one embodiment, the protein binder is an antibody comprising the variable domains or CDRs of 4H8-E3.

According to one embodiment of the invention the protein binder comprises:
a) a pair of heavy/light chain complementarity determining regions (CDRs) comprised in the heavy/light chain variable region sequence pairs set forth in SEQ ID NOS: 33 and 40;
b) comprising a set of heavy/light chain complementarity determining regions (CDRs) comprising the following sequences: HC CDR1 (SEQ ID NO: 34 or 37)
- HC CDR2 (SEQ ID NO: 35 or 38)
- HC CDR3 (SEQ ID NO: 36 or 39)
- LC CDR1 (SEQ ID NO: 41 or 44)
- LC CDR2 (SEQ ID NO: 42 or 45), and - LC CDR3 (SEQ ID NO: 43 or 46),
c) the heavy/light chain complementarity determining regions (CDRs) of b), wherein at least one of the CDRs has up to 3 amino acid substitutions relative to each SEQ ID NO: 34-39 or 41-46; have
d) comprising the heavy/light chain complementarity determining regions (CDRs) of b) or c) such that at least one of the CDRs is 66% or greater relative to SEQ ID NOS: 34-39 or 41-46, respectively; having sequence identity,
The CDRs are embedded in suitable protein frameworks so that they can bind human iRhom2 with sufficient binding affinity and inhibit or reduce TACE/ADAM17 activity.

These CDRs are the CDR set of antibody 4H8-E3 and are determined by different approaches (SEQ ID NOs: 34-39 are determined by the paratome CDR identification procedure (http://ofranceservices.biu.ac.il/site/ services/paratome), and SEQ ID NOS: 41-46 are determined by in-house methods).

As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen binding sites found within the variable regions of both heavy and light chain polypeptides. ing. These particular regions have been described by Kabat et al. (1977), Kabat et al. (1991), Chothia et al. (1987) and MacCallum et al. (1996), where the definitions are, when compared to each other, amino acid residues contains duplicates or subsets of

Nonetheless, application of any definition that refers to CDRs of antibodies or grafted antibodies or variants thereof is intended to be within the terms defined and used herein. The amino acid residues encompassing the CDRs defined by each of the above references are shown in Table 1 below for comparison. Note that this numbering may differ from the actual CDRs disclosed in the enclosed Sequence Listing, as CDR definitions vary from case to case.

As used herein, the term "framework" when used in reference to an antibody variable region is entered to mean all amino acid residues outside the CDR regions within the variable region of an antibody. Thus, the variable region framework is about 100-120 amino acids long, but is intended to refer only to amino acids other than the CDRs.

As used herein, the term "capable of binding target X with sufficient binding affinity" means that each binding domain binds the target with a _KD of ^10-4 or less. must be understood as K _D is the equilibrium dissociation constant, which is the ratio k _off /k _on between a protein binder and its antigen. K _D and affinity are inversely related. Since the K _D value is related to the concentration of protein binder (the amount of protein binder required for a particular experiment), the lower the K _D value (the lower the concentration), the higher the affinity of the binding domain. The table below shows monoclonal identities with typical K _D ranges.

Preferably the protein binder has up to 2 amino acid substitutions, more preferably up to 1 amino acid substitution

Preferably, at least one of the CDRs is ≧67%; ≧68%; ≧69%; ≧70%; ≧71%; ≧72%; ≧76%; ≧77%; ≧78%; ≧79%; ≧80%; ≧81%; ≧82%; ≧88%; ≧89%; ≧90%; ≧91%; ≧92%; ≧93%; ≧94%; ≧95%; and most preferably ≧100%.

The term "% sequence identity" as used herein should be understood as follows: the two sequences being compared are aligned to give the maximum correlation between the sequences. This may involve inserting "gaps" in one or both sequences to enhance the degree of alignment. Percent identity can then be determined over the entire length of each sequence compared (a so-called global alignment), which can be, inter alia, the same or similar in length or short defined in length. alignments (so-called local alignments).

In the above context, an amino acid sequence that has at least, e.g., 95% "sequence identity" to a query amino acid sequence is one in which the sequence of the subject amino acid sequence contains at most 5 amino acid changes per 100 amino acids each of the query amino acid sequence. It is intended to mean that the sequence of the subject amino acid sequence is identical to the query sequence, except that it may contain. In other words, to obtain an amino acid sequence having at least 95% sequence identity to a query amino acid sequence, up to 5% (5 out of 100) of the amino acid residues in the subject amino acid sequence must be replaced with another amino acid. may be inserted or substituted with or deleted. Methods to compare the identity and homology of two or more sequences are well known in the art.

The percentage identity of two sequences can be determined, for example, using a mathematical algorithm. Preferred examples of mathematical algorithms include, but are not limited to, the BLAST or NBLAST programs and the BLAST family of programs such as FASTA. Sequences with some degree of identity to other sequences can be identified by these programs.

In addition, Wisconsin sequence analysis packages such as program BESTFIT and program 9.1 available in GAP can be used to determine percent identity between two polypeptide sequences. When referred to herein are amino acid sequences sharing a certain degree of sequence identity to a reference sequence, the sequence differences are preferably due to conservative amino acid substitutions. Preferably, such sequences retain the activity of the reference sequence, albeit at a slower rate, for example.

Preferably, at least one of the CDRs has been subjected to CDR sequence modifications including:
● Affinity maturation ● Reduced immunogenicity

Affinity maturation in the process of increasing the affinity of a given antibody in vitro Like its natural counterpart, in vitro affinity maturation is based on the principle of mutation and selection. It has been successfully used to optimize other peptide molecules such as antibodies, antibody fragments or antibody mimetics. Random mutations within CDRs are introduced using radiation, chemical mutagen or error-prone PCR. Furthermore, genetic diversity can be increased by chain shuffling. A few rounds of mutation and selection using a display method such as phage display typically yield antibody fragments with affinities in the low nanomolar range. For principles, see Eylenstein et al. (2016). The contents of which are incorporated herein by reference.

Humanized antibodies comprise the CDR regions from murine sequences with the necessary framework backmutations into the V regions from human sequences. Thus, the CDRs themselves can provoke an immunogenic response when a humanized antibody is administered to a patient. Methods for reducing immunogenicity caused by CDRs are disclosed in Harding et al. (2010), the contents of which are incorporated herein by reference.

According to one embodiment of the invention the framework is a human VH/VL framework. VH stands for heavy chain variable domain and VL for light chain variable domain (kappa or lambda) of IgG-type antibodies.

According to one embodiment of the invention, the protein binder comprises a) a heavy/light chain variable domain (VD)
- HC VD (SEQ ID NO: 33), and - LC VD (SEQ ID NO: 40).
b) the heavy/light chain variable domains (VDs) of a), provided that: ● the HC VDs have at least 80% sequence identity with their respective SEQ ID NO: 33; and/or ● the LC VDs are their respective SEQ ID NO: 40 c) the heavy/light chain variable domains (VDs) of a) or b) having ≧80% sequence identity with the respective SEQ ID NO: A heavy/light chain variable domain (VD) with up to 10 amino acid substitutions for 33 and/or 40;
Said protein binder binds human iRhom2 with sufficient binding affinity and is still able to inhibit or reduce TACE/ADAM17 activity.

Preferably, the HC VD and/or LC VD are ≧81%; ≧82%; ≧83%; ≧84%; ≧85%; ≧86%; ≧89%; ≧90%; ≧91%; ≧92%; ≧93%; ≧94%; ≧95%; Has 100% sequence identity.

A "variable domain", when used in reference to an antibody or heavy or light chain thereof, is intended to mean that part of an antibody that confers antigen binding on the molecule and is not the constant region. The term is intended to include functional fragments thereof that retain part of the binding function of the entire variable region. Variable region-binding fragments include, for example, functional fragments such as Fab, F(ab) ₂ , Fv, and single-chain Fv (scfv). Such functional fragments are well known to those skilled in the art. Thus, the use of these terms in describing functional fragments of heteromeric variable regions is intended to correspond to definitions well known to those of ordinary skill in the art. Such terms are described, for example, in Huston et al. (1993) or Pluckthun and Skerra (1990).

According to one embodiment of the invention, at least one amino acid substitution discussed above is a conservative amino acid substitution.

"Conservative amino acid substitutions" have less effect on protein binder function than non-conservative substitutions. Although there are many ways to classify amino acids, they are often classified into six major groups based on their structure and the general chemical properties of the R groups.

In one embodiment, a "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with the following side chains:
- Basic side chains (e.g. lysine, arginine, histidine)
● Acidic side chains (aspartic acid, glutamic acid, etc.)
Uncharged polar side chains (glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, etc.)
Nonpolar side chains (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, etc.)
β-branched side chains (threonine, valine, isoleucine, etc.)
Aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine)

Other conservative amino acid substitutions can also occur across amino acid side chain families, such as replacing asparagine with aspartic acid to modify the charge of a peptide. Thus, for example, a predicted nonessential amino acid residue in an HR domain polypeptide may cross-family another amino acid residue from the same side chain family or homologue (e.g., asparagine for aspartic acid, glutamine for glutamic acid, ) is preferably replaced with Conservative changes can also include substitution of chemically homologous non-natural amino acids (ie, synthetic non-natural hydrophobic amino acids for leucine, synthetic non-natural aromatic amino acids for tryptophan).

According to one embodiment of the invention, the protein binder has at least one of:
- A target binding affinity as measured by SPR of ≥50% for iRhom2 when compared to the protein binder described above, and/or - ≥50% of the inhibitory or reducing effect on TACE/ADAM17 activity of the protein binder described above. .

As used herein, the term "binding affinity" is intended to mean the strength of a binding interaction and thus includes both actual as well as apparent binding affinity. The actual binding affinity is the ratio of association rate to dissociation rate. Thus, conferring or optimizing binding affinity involves altering either or both of these components to achieve the desired level of binding affinity. Apparent affinities can include, for example, the avidity of interactions. For example, bivalent heteromeric variable region binding fragments can exhibit altered or optimized binding affinities due to their valency.

A suitable method for measuring binding agent affinity is through surface plasmon resonance (SPR). This method is based on the phenomenon that occurs when surface plasmon waves are excited at the metal/liquid interface. Light is projected onto the side of the surface not in contact with the sample and reflected, and SPR causes a drop in reflected light intensity at certain combinations of angle and wavelength. A biomolecular binding event causes a change in refractive index at the surface layer, which is detected as a change in the SPR signal.

A binding event can be either binding association or dissociation between a receptor-ligand pair. Changes in refractive index can be measured essentially instantaneously, thus allowing determination of the individual components of the affinity constant. Specifically, it enables accurate measurement of association rate (k _on ) and dissociation rate (k _off ).

Measuring k _on and k _off is useful because it allows identification of altered or optimized variable regions. For example, a modified variable region, or heteromeric binding fragment thereof, may be more effective, eg, because it has a higher k _on compared to a variable region and heteromeric binding fragment that exhibit similar binding affinities. A molecule with a higher k _on can specifically bind and inhibit its target at a faster rate, resulting in increased efficacy. Likewise, molecules of the invention may be more effective because they exhibit lower _koff values compared to molecules with similar binding affinities. Molecules with lower k _off rates are more potent because, once bound, the molecule dissociates from its target more slowly. While reference is made to modified and optimized variable regions of the invention, including heteromeric variable region binding fragments thereof, the above methods for measuring association and dissociation rates are not useful for therapeutic or diagnostic purposes. It can be applied to essentially any protein binder or fragment thereof to identify more effective binding agents.

Methods of measuring affinity, including association and dissociation rates using surface plasmon resonance are well known in the art and can be described, for example, in Jonsson and Malmquist, (1992) and Wu et al. (1998). . Additionally, one instrument well known in the art for measuring binding interactions is the BIAcore 2000 instrument commercially available through Pharmacia Biosensors (Uppsala, Sweden).

Preferably, the target binding affinity is ≧51%, ≧52%, ≧53%, ≧54%, ≧55%, ≧56%, ≧57%, ≧58%, ≧59%, ≧60% of the reference binding agent. %, ≧61%, ≧62%, ≧63%, ≧64%, ≧65%, ≧66%, ≧67%, ≧68%, ≧69%, ≧70%, ≧71%, ≧72%, ≧73%, ≧74%, ≧75%, ≧76%, ≧77%, ≧78%, ≧79%, ≧80%, ≧81%, ≧82%, ≧83%, ≧84%, ≧85 %, ≧86%, ≧87%, ≧88%, ≧89%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, most preferably ≧99%.

As used herein, quantification of inhibitory or reducing effects on TACE/ADAM17 activity can be performed in comparison to benchmark binding agents, e.g., in respective TNF shedding assays (e.g., Figure 2 and Example 5).

According to another aspect of the invention there is provided a protein binding agent that competes for binding of human iRhom2 with any of the protein binders described above.

As regards the format or structure of such protein binders, the same preferred embodiments as above apply. In one embodiment, said protein binder is a monoclonal antibody, or a target-binding fragment or derivative thereof that retains target-binding ability, or an antibody mimetic.

As used herein, the term "compete for binding" is used with reference to one of the antibodies defined by said sequence, which, like said sequence-defined protein binder, actually are active binding to the same target, or target epitope or domain or subdomain, and are variants of the latter. The efficiency (eg, kinetics or thermodynamics) of binding can be the same as, greater than, or less than the efficiency of the latter. For example, the equilibrium binding constants for binding to substrate can differ for two antibodies.

Such competition for binding can be suitably measured in competitive binding assays. Such assays are described by Finco et al. 2011, the contents of which are incorporated herein by reference, and their meanings for claim interpretation are disclosed in Deng et al., 2018, the contents of which are incorporated herein by reference. be done.

According to another aspect of the invention there is provided a protein binder that binds essentially the same or to the same epitope on iRhom2 as the protein binder described above.

Suitable epitope mapping techniques are available to test this property, including, among others, the following:
●X-ray co-crystal analysis and cryo-electron microscopy (cryo-EM)
● Array-based oligo-peptide scanning ● Site-directed mutagenesis mapping ● High-throughput shotgun mutagenesis epitope mapping ● Hydrogen-deuterium exchange ● Cross-linking mass spectrometry

These methods are disclosed and discussed, inter alia, in Banik et al. (2010) and DeLisser (1999), the contents of which are incorporated herein by reference.

According to another aspect of the invention, a nucleic acid molecule encoding a binding agent according to any one of the preceding claims.

A given sequence of an encoded binding agent may have different sequences such nucleic acid molecules due to the degeneracy of the genetic code.

Such nucleic acid molecules can be used for pharmaceutical purposes. In such cases, it is the RNA-derived molecule that is administered to the patient, where the patient's protein expression machinery expresses the respective binding agent. The mRNA can, for example, be delivered in suitable liposomes and contain either specific sequences or modified uridine nucleosides to evade immune response and/or improve folding and translation efficiency, sometimes including Include cap modifications at the 5' and/or 3' ends to target to specific cell types.

Such nucleic acid molecules can be used to transfect an expression host and then express the actual binding agent. In such cases, the molecule may be a cDNA, optionally incorporated into an appropriate vector.

According to another aspect of the invention, the use of a protein binder as described above comprises
For the treatment of a human or animal subject ■ who has been diagnosed with an inflammatory disease, ■ who is suffering from an inflammatory disease, or who is at risk of developing an inflammatory disease, or who is at risk of preventing such a disease. provided (for the manufacture of medicines) to

According to another aspect of the invention there is provided a pharmaceutical composition comprising a protein binder as described above and optionally one or more pharmaceutically acceptable excipients.

According to another aspect of the invention, a combination comprises (i) a protein binder as defined above, or a pharmaceutical composition as defined above, and (ii) one or more therapeutically active compounds.

According to another aspect of the invention, there is provided a method for treating or preventing an inflammatory condition, comprising (i) a protein binder as described above, (ii) a pharmaceutical composition as described above, or (iii) a pharmaceutical composition as described above. It includes administration of a therapeutically sufficient amount of the described combination to a human or animal subject.

According to another aspect of the invention, there is provided a therapeutic kit of parts, comprising:
a) a composition as defined above, a pharmaceutical composition as defined above, or a combination as defined above;
b) a device for administering the composition, composition or combination, and c) instructions for use.

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive, the invention being disclosed It is not limited to the embodiment. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" excludes the plural. not something to do. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

All amino acid sequences disclosed herein are written from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are written 5'->3'.

Example 1: Generation of Peptides for Immunization and Peptide Binding ELISA Analysis

Peptides were synthesized using a parallel peptide synthesizer (peptides 1-5, 7-9 and 1b-3b; MultiPep RSi, Intavis AG, Germany), a microwave peptide synthesizer (peptide 6; Liberty Blue, CEM, USA), or a fluorenyl Synthesized on a custom continuous flow peptide synthesizer (peptides 10, 11 and 4b) using methoxycarbonyl (Fmoc)-based solid-phase peptide synthesis. [Chan, WC, White, PD Solid Phase Peptide Synthesis, A Practical Approach (Oxford University Press Inc., New York, 2000].

The sequence was assembled stepwise from C to N-terminus using Fmoc-protected L-amino acids with side chain protecting groups. Once the tethered assembly peptide was complete, it was cleaved from the resin with 95% TFA, 4% triethylsilane and 1% water. The crude product was dissolved in 15% acetonitrile in 0.1% aq TFA and purified by reverse-phase HPLC using an Orbit C18, 10 μm, 100 viti column (MZ Analysentechnik, Germany). The resulting purified fractions were analyzed by analytical HPLC using a Kinetex EVO C18, 5 μm, 100 Asahi column (Phenomenex, USA) and MALDI TOF mass spectrometry (Bruker, USA, Ultraflex III). Fractions were lyophilized to obtain the corresponding TFA salts.

For peptides 10 and 11, linear peptides identified by mass spectrometry were oxidized to the corresponding cyclic disulfides by DMSO-mediated oxidation. For this purpose the linear peptide was dissolved in 5% acetic acid at a concentration of 1 mg/ml. The pH was adjusted to 6 with (NH4)2CO3 and DMSO was added to a final concentration of 10-20%. Oxidation was allowed to proceed for 24 hours at room temperature. The reaction mixture was then diluted with solvent A and the product was purified on a reverse phase C18 column and analyzed as above. Fractions containing disulfide-cyclized peptides were pooled and lyophilized. [Chan, W.C. and White, P.D., Fmoc Solid Phase Peptide Synthesis, A Practical Approach (Oxford University Press Inc., New York, 2000, Chapter 3.3, page 97]

KLH conjugation was performed using pre-activated KLH (Imcejt™ Maleimide Activated mcKLH, Thermo Scientific, USA). Briefly, mcKLH was dissolved in ultrapure water at a concentration of 10 mg/ml. The desired peptide was added to Imject ^™ Maleimide Conjugation Buffer (Thermo Scientific, USA) at a concentration of 5 mg/mL when 8 M urea (pH 7.2) was added to dissolve the peptide. Dissolved. The peptide solution was mixed with the mcKLH solution and incubated at room temperature for 2-6 hours. The mixture was dialyzed overnight against 400 mL of PBS against 3500 MW cut-off (MWCO) dialysis tubing. After dialysis, the mixture was diluted with PBS to obtain the desired concentration.

Biotinylation was performed with α-biotin-ω-maleimide undeca(ethylene glycol) (biotin-PEG(11)-mal). Peptides were dissolved in PBS pH 7,4. If necessary, acetonitrile was added to dissolve the peptide. Biotin-PEG(11)-mal was dissolved in DMF and added to the peptide solution (weight = 1:1). The reaction was run overnight and then purified on a reverse phase C18 column and analyzed as above.

FIG. 1 shows the peptides used for immunization and/or peptide binding ELISA analysis, NCBI reference sequences NM_024599.5, NP_078875.4 for human iRhom2 and NCBI reference sequence NM_022450.3 for human iRhom1. , NP_071895.3. and indicates its name, position and amino acid. Terminal cysteine residues added to all peptides except peptides 6 and 7 for coupling to KLH (for immunization) and/or biotin (for peptide binding ELISA analysis) are exemplified by "-C-". Internal cysteine residues are replaced with alpha-aminobutyric acid (Abu) where indicated. Peptides 1-4 correspond to the amino acids of TMD1 (highlighted in italics) and the adjacent extracellular juxtamembrane region of human iRhom2 (FIG. 1A). Peptides 5-7 resemble segments within the large extracellular loop 1 of human iRhom2 that connects TMD1 and TMD2 (Fig. 1B). Peptides 8-11 represent the amino acids of TMD7 (highlighted in italics) and the adjacent C-terminal tail of human iRhom2 (Fig. 1C). Peptides 1b-4b are human iRhom1 homologues of peptides 1-4 and thus correspond to the amino acids of TMD1 (highlighted in italics) and the adjacent extracellular juxtamembrane region of human iRhom1 (FIG. 1D).

Example 2: Breeding of iRhom2 Knockout Mice for Immunization

The human versus mouse iRhom2 protein has high sequence homology (see NCBI reference sequence NP_078875.4 for human iRhom2 and NCBI reference sequence NP_766160.2 for mouse iRhom2), and extracellular loops 1, 2 of human versus iRhom2 , 3 and C-terminal tail amino acid sequence identities calculated to be 89.96%, 100.00%, 100.00% and 96.97%, respectively), of immunized iRhom2 knockout but not wild-type mice. bred for

In summary, the Rhbdf2tm1b (KOMP) Wtsi mouse strain (Rhbdf2 is an alternative name for iRhom2) was designated for resuscitation from the KOMP Mouse Biology Program at the University of California, Davis, and three heterozygous male mice are available. It became so. These three on the C57BL/6N background (C57BL/6N-Rhbdf2tm1b(KOMP) Wtsi) were mated with wild-type female mice on the 129Sv/J genetic background to generate heterozygous offspring. These heterozygous mice were bred with each other to generate male and female mice with a zygotic knockout of the Rhbdf2 gene. The resulting conjugative Rhbdf2 knockout mouse colony was further expanded for immunization.

Example 3: Immunization of mice and serum titer analysis

Three cohorts of 8-10 week old male and female iRhom2 knockout mice (described in Example 2) were immunized with peptide mixtures A, B and C, respectively. Mixture A consisted of equal amounts of the four keyhole limpet hemocyanin (KLH) binding peptides 1, 2, 3 and 4. Mixture B was composed of equal amounts of the three KLH-binding peptides 5, 6 and 7 and mixture C was composed of equal amounts of the four KLH-binding peptides 8, 9, 10 and 11. 50 μg of the peptide mixture was emulsified with 20 μl of GERBU adjuvant MM ^™ (GERBU Biotechnik, Germany), adjusted with 10 mM HEPES buffer (PH 7.6) and injected intraperitoneally (in a final volume of 100 μl per injection per mouse). IP) was administered. Groups of 10 mice were injected 5 times every 10 days. Ten days after the fifth injection, blood (serum) was collected and tested for antibody titer.

Evaluation of immune response was performed by serum antibody titer analysis applying ELISA and FACS methods. For FACS analysis, serum diluted 1:50 in PBS containing 3% FBS was treated with goat F(ab′)2 anti-mouse IgG (H+L)-R-phycoerythrin (RPE) conjugate (Gianova, Germany) as secondary antibody. ) was used in mouse L929 cells stably expressing human iRhom2. Parental L929 cells were used as a negative control. The test was performed on an Accuri C6 Plus (BD Biosciences, USA) flow cytometer. Pre-immune serum (“PIS”) collected on day 0 of the immunization protocol served as a negative control.

Complementarily, whole-head immune sera were tested by enzyme-linked immunosorbent assay (ELISA): sera were diluted 1:500, 1:2500 and 1:12,500 in PBS containing 1% BSA and added to horseradish. Binding to plates coated with 1 μg/ml of each biotinylated peptide mixture was examined by detection with a peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (Southern Biotech, USA). An irrelevant protein (BSA) and pre-immune serum collected on day 0 of the immunization protocol served as negative controls.

For further boosting of the immune response, immunization with the peptide mixture was extended to 4 days after serum collection with another 2 injections every 2 weeks, followed by a 10 day boost. Spleens of selected animals were harvested 4 days after the final boost, lymphocytes were isolated and cryopreserved for subsequent fusion.

Example 4: Collection and Fusion of Lymphocytes for Generation of Hybridomas

Cryopreserved splenic lymphocytes from three selected animals per immunization cohort were thawed and group-specifically fused with Ag8 mouse myeloma cells for the generation of hybridoma cells. Fused cells were plated and grown on 96-well plates in the presence of hypoxanthine-aminopterin-thymidine (HAT) medium. Group-specific fusions allowed retrospective assignment of emerging hybridomas to their respective immunization groups.

Example 5: Screening of Hybridoma Supernatants for Candidate Selection

After 14 days of culture, hybridoma cell supernatants were harvested and subjected to ELISA-based functional screening for iRhom2 activity-neutralizing antibodies instead of selecting for iRhom2 binding antibodies. The critical role of iRhom2 in the TACE-mediated release of tumor necrosis factor alpha (TNFα) from macrophages is well established (McIlwein et al., 2012, Adrain et al., 2012, Siggs et al., 2012), so the human TNF-α DuoSet An ELISA (R&D Systems, USA) was employed to detect lipopolysaccharide (LPS)-induced release of endogenous TNFα from human THP-1 macrophage cells in the presence and absence of all 5280 peptide immunization-derived hybridoma supernatants. compared.

Briefly, on day 1, Nunc black Maxisorp® 96-well plates (Thermo Fisher Scientific, USA) were treated with 4 μg/ml TBS with a mouse anti-human TNFα capture antibody (provided as part of the DuoSet ELISA kit). filled) with 100 μl/well overnight at 4°C. On day two, the capture antibody solution was removed and the Maxisorp® plates were blocked with 300 μl/well TBS, 1% BSA overnight at 4°C. On day 3, 20,000 THP-1 (US-type culture harvest) cells in 80 μl of normal growth medium were seeded into each well of a Greiner CELLSTAR V-bottom 96-well plate (Thermo Fisher Scientific, USA), and Pre-incubated with 20 μl of hybridoma supernatant for 30 minutes at 37° C., 5% CO ₂ . In the control group, 20 μl of standard growth medium was added instead of the hybridoma supernatant. Cells (except unstimulated controls) were then plated with LPS (Sigma-Aldrich, USA) in 300 ng/ml growth medium at 20 μl per well to a final concentration of 50 ng/ml at 37° C., 5% CO ₂ . Stimulated for 2 hours. The 96-well plate was then centrifuged to pellet the cells. In parallel, the blocking buffer was removed from the MaxiSorp® plates and the plates were washed 4 times with 350 μl per well of TBS-T (Karl Roth, Germany) on a 96-head plate washer (Tecan Group, Switzerland). washed twice. To avoid drying, 30 μl of TBS was immediately added to each well of the MaxiSorp® plate before transferring 70 μl of cell-free supernatant per sample.

In addition, 100 μl of recombinant human TNFα protein (provided as part of the DuoSet ELISA kit) diluted in TBS to defined concentrations was added to the plate as a standard reference. 100 μl per well of biotinylated goat anti-human TNFα detection antibody (provided as part of the DuoSet ELISA kit) in 50 ng/ml TBS was then added, protected from direct light, and the plates were incubated for 2 hours at room temperature. After 4 washes with 350 μl per well of TBS-T (Carl Roth, Germany) on a 96-head plate washer (Tecan Group, Carl Roth, Switzerland), carefully removing all traces of buffer from the 4th cycle onwards. , 100 μl of streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS was added to each well, again protected from direct light and the plate was incubated for 30 minutes at room temperature. Wash again 4 times with 350 μl per well of TBS-T (Carl Roth, Germany) on a 96-head plate washer (Tecan Group, Switzerland), carefully remove all traces of buffer from the 4th cycle onwards, then place at room temperature. 100 μl of AttoPhos substrate solution (Promega, USA) was added for 1 hour incubation in the dark. Fluorescence of each well was collected with an excitation wavelength of 435 nm and an emission wavelength of 555 nm using an infinite M1000 PRO (Tecan Group, Switzerland) microplate reader.

FIG. 2 shows representative results of these experiments on one 96-well plate showing the effect of hybridoma supernatants derived from peptide immunizations on LPS-induced release of TNFα from THP-1 cells. Out of a total of 5280 hybridoma supernatants tested, the supernatant taken from the hybridoma cell population on plate number 4, column H, row 8 (4H8) reveals LPS-induced TNFα shedding in THP-1 cells. It's the only thing that interferes.

Example 6: Subcloning of hybridoma cell population 4H8

Since the hybridoma cell population 4H8 appeared to be of oligoclonal origin, subcloning was performed applying the classical broth dilution method to isolate a monoclonal hybridoma cell pool.

Briefly, cells of hybridoma population 4H8 were counted and dilution factors calculated to end up with an average of 2 cells per well of a 96-well plate. Cells were diluted accordingly and wells with growing single cell populations were identified through a microscope. After expanding these monoclonal hybridoma populations for approximately 3 weeks, supernatants were collected and compared for inhibitory effects on LPS-induced release of TNFα from THP-1 cells as described in Example 5. Three 4H8 subclones, designated 4H8-D4, 4H8-E3 and 4H8-G8, were found to significantly inhibit TNFα shedding and were therefore expanded and pooled.

Example 7: Purification of Antibody from Hybridoma Subclone 4H8-E3

This example describes the purification of antibodies from the supernatant of hybridoma subclone 4H8-E3 using affinity chromatography.

In summary, Protein G Sepharose is primarily recommended for immobilizing IgG antibodies and described as less suitable for binding IgM antibodies, whereas Protein G Sepharose columns give good yields of both antibody isotypes. It was empirically found to result in Therefore, supernatants collected from hybridoma subclone 4H8-E3 were pooled and loaded onto an equilibrated Protein G Sepharose pre-packed gravity flow column (Protein G GraviTrap ^™ , GE Healthcare, UK) for antibody capture. The column was then washed once with binding buffer and captured antibody was eluted with elution buffer (both buffers are provided as part of the Ab Buffer Kit; GE Healthcare, UK). The eluate fraction was then desalted using PD Miditrap G-25 columns (GE Healthcare, UK) and filtered through Amicon Ultra-4 Centrifugal Filter Units (Sigma-Aldrich, USA) with a cutoff of 30 kDa. Purified samples were concentrated via Finally, the concentration of purified protein was measured using a NanoDrop 2000/c spectrophotometer (Thermo Fisher Scientific, USA).

Example 8: Isotyping of the antibody 4H8-E3 of the invention

As a next step, a mouse IgG/IgM ELISA was performed to determine the isotype of the purified antibody 4H8-E3 of the invention. Briefly, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were loaded with 1 μg/ml TBS, goat anti-mouse IgG+IgM (H+L) capture antibody (US Science-Aldrich) 100 μl/well. and coated overnight at 4°C. On day 2, the capture antibody solution was removed and the Maxisorp® plates were blocked with 300 μl per well of Pierce protein-free (TBS) blocking buffer (Thermo Fisher Scientific, USA) for 1 hour at room temperature. The blocking buffer was then removed and the plates were washed 3 times with 350 μl per well of TBS-T (Karl Roth, Germany) on a 96-head plate washer (Tecan Group, Switzerland). followed by mouse IgG (Thermo Fisher Scientific, USA) and mouse IgM (Sigma-Aldrich, USA) antibodies (both starting at 1 μg/ml TBS) in 100 μl TBS per well as blanks and negative controls, standard reference mouse IgG (Thermo Fisher Scientific, USA) and mouse IgM (Sigma-Aldrich, USA) antibodies (100 μl per well of 3 μg/ml TBS as positive and specificity controls, respectively, and the present invention at 3 μg/ml TBS) of purified antibody 4H8-E3 was added to the wells and incubated for 2 hours at room temperature, then TBS-T (Karl Roth, Germany) at 350 μl per well on a 96-headplate washer (Tecan Group, Switzerland). Plates were washed again three times.

For isotype detection, half of the samples were protected from direct light and treated with AP-conjugated goat anti-mouse IgM (Sigma-Aldrich, USA) or AP-conjugated goat anti-mouse IgG F(ab′)2 fragment (Gianova, Germany) detection antibodies. was incubated with 100 μl/well diluted 1:5000 in TBS for 1.5 hours at room temperature. TBS-T (Carl Roth, Germany) was washed another 3 times with 350 μl per well, carefully removing all traces of buffer after the 3rd cycle using a 96-head plate washer (Tecan Group, Switzerland). Afterwards, 100 μl of AttoPhos substrate solution (Promega, USA) was added and incubated in the dark and at room temperature for 10 minutes. Fluorescence of each well was collected with an excitation wavelength of 435 nm and an emission wavelength of 555 nm using an infinite M1000 PRO (Tecan Group, Switzerland) microplate reader.

Figure 3 clearly shows that the antibody 4H8-E3 of the invention is of the mouse IgM isotype.

Example 9: Determination of Target Region Recognized by Antibody 4H8-E3 of the Invention

A peptide binding ELISA assay was then performed to show that the purified antibody 4H8-E3 of the invention recognizes any of the peptides administered to these animals from which the hybridoma clone 4H8 was derived, thereby recognizing the antibody of the invention. It was verified whether it emits light on the target area recognized by 4H8-E3.

Briefly, on day 1, Nunc black Maxisorp® 96-well plates (Thermo Fisher Scientific, USA) were added at 4° C. with 100 μl per well of peptides 1-11, peptides 1-4 (mixture A). , 5-7 (mixture B), and 8-11 (mixture C) were each coated overnight with 10 μg/ml TBS (thus the final concentration of each peptide in mixtures 1-4 and 8-11 was , 2.5 μg/ml vs. 3.3 μg/ml in mixtures 5-7). On day 2, the peptide solution was removed and the Maxisorp® plates were blocked with 300 μl per well of Pierce protein-free (TBS) blocking buffer (Thermo Fisher Scientific, USA) for 1.5 hours. The blocking buffer was then removed and the plates were washed 4 times with 350 μl per well of TBS-T (Karl Roth, Germany) on a 96-head plate washer (Tecan Group, Switzerland).

Then 100 μl per well of TBS as blank control, mouse anti-biotin immunization (clone BN-34, Sigma-Aldrich, USA) with 0.3 μg/ml TBS as coating control, purified antibody of the present invention with 3 μg/ml TBS 4H8-E3, and an isotype control mouse IgM immunization (clone MOPC 104E, Sigma-Aldrich, USA) to the purified antibody 4H8-E3 of the invention at 3 μg/ml TBS was pretreated with single peptides 1 to 11 or a mixture of each. Added to plated wells and incubated at room temperature for 4 hours. The plate was then washed again on a 96-head plate washer (Tecan Group, Switzerland) 4 times with 350 μl per well of TBS-T (Karl Roth, Germany), protected from direct light, and washed 1:2000 in TBS. 100 μl/well of AP-conjugated goat anti-mouse IgG/IgG/IgM F(ab′)2 fragment (Sigma-Aldrich, USA) diluted to 100 μl/well for 1 hour at room temperature. After 4 washing steps with 350 μl per well of TBS-T (Carl Roth, Germany) in a 96-head plate washer (Tecan Group, Switzerland), carefully removing all traces of buffer from the 4th cycle onwards, 100 μl of AttoPhos substrate solution (Promega, USA) was added and incubated for 1 hour in the dark and at room temperature. Fluorescence of each well was collected with an excitation wavelength of 435 nm and an emission wavelength of 555 nm using an infinite M1000 PRO (Tecan Group, Switzerland) microplate reader.

Figure 4 shows representative results of this experiment. Coating controls confirm the abundance of immobilized biotinylated peptides individually or as peptide mixtures (FIGS. 4A, C, E). Consistent with clone 4H8, derived from mice immunized with a mixture of peptides 1-4 (mixture A), the antibody 4H8-E3 of the invention was coated with these peptides individually or as a mixture. to peptides 5, 6 and 7, which resemble various segments of the large extracellular loop (Fig. 4D), regardless of whether the does not show the binding of In contrast, strong binding of the antibody 4H8-E3 of the invention to mixed A consisting of peptides 1, 2, 3, and 4 as well as the single peptide 3 was shown (Fig. 4B) and recognized by the antibody 4H8-E3 of the invention. The epitope identified was found to be localized within amino acids 431-459 of the extracellular juxtamembrane domain of human iRhom2. Data for antibody 4H8-E3 of the invention are shown after normalization to IgM isotype controls.

Example 10: Assessment of binding specificity of antibody 4H8-E3 of the invention

To address the specificity of the purified antibody 4H8-E3 of the present invention, i.e., whether this antibody specifically recognizes the specific peptide peptide 3, which resembles the TMD1-flanked extracellular juxtamembrane region of human iRhom2. , or to question whether the antibody 4H8-E3 of the invention also binds to peptides that reflect the homologous regions of the closely related family member human iRhom1, another series of peptide binding ELISA experiments was conducted. It was implemented.

Briefly, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of single biotinylated peptides 1-4; Mixture A consisting of ~4, single biotinylated peptides 1b-4b, and mixture D consisting of peptides 1b-4b were each added to 10 μg/ml PBS (so the final concentration of each peptide in both mixtures was 2.0 μg/ml). was 5 μg/ml) overnight at 4° C. On day 2, the peptide solution was removed and the MaxiSorp® plates were placed in Pierce protein-free (TBS) blocking buffer (Thermo Fisher) at room temperature. Scientific, USA) was blocked for 1.5 hours with 300 μl per well.

Blocking buffer was then removed and plates were washed 4 times with 350 μl per well of PBS-T (Karl Roth, Germany) on a 96-head plate washer (Tecan Group, Switzerland). Then 100 μl per well of PBS as a blank control, 0.3 μg/ml mouse anti-biotin antibody (clone BN-34, Sigma) in PBS as a coating control, 3 μg/ml PBS of the purified antibody of the invention 4H8-E3 as a coating control, and Mouse IgM antibody (clone PFR-03, Sigma) in PBS 3 μg/ml was added to wells precoated with single peptides 1-4, 1b-4b or a mixture of each as an isotype control for the purified antibody 4H8-E3 of the invention. , incubated for 4 hours at room temperature. Plates were then washed again 4 times on a 96-head plate washer (Tecan Group, Switzerland) with 350 μl per well of PBS-T (Carlos, Germany) and diluted 1:2000 with PBS, avoiding direct light. Incubated with 100 μl/well of AP-conjugated goat anti-mouse IgG/IgG/IgM F(ab′)2 fragment (Sigma-Aldrich, USA) for 1 hour at room temperature.

After 4 washing steps of 350 μl per well of PBS-T (Carl Roth, Germany) were passed again through the 96-head plate washer (Tecan Group, Switzerland), carefully removing all traces of buffer from the 4th cycle onwards. , 100 μl of AttoPhos substrate solution (Promega, USA) was added and incubated for 1 hour in the dark and at room temperature. Fluorescence of each well was collected with an excitation wavelength of 435 nm and an emission wavelength of 555 nm using an infinite M1000 PRO (Tecan Group, Switzerland) microplate reader.

Figure 5 shows representative results of this experiment. Coating controls again confirmed the abundance of immobilized biotinylated peptides, either individually or as peptide mixtures (Fig. 5A,C), from peptides 1, 2, 3 and 4 of antibody 4H8-E3 of the invention. binding to a single peptide 3 resembling amino acids 431-459 of the extracellular juxtamembrane domain of human iRhom2 (Fig. 5B). In contrast, the antibody 4H8-E3 of the present invention consists of or individually coated mixtures of peptides 1b, 2b, 3b and 4b that reflect homologous amino acid sequences within the related family member human iRhom1 (Fig. 5D). D, providing evidence that the antibody 4H8-E3 of the invention specifically binds to human iRhom2 and therefore does not recognize the homologous portion in human iRhom1. Data for antibody 4H8-E3 of the invention are shown after normalization to IgM isotype controls.

Example 11: Analysis of the inhibitory effect of the antibody 4H8-E3 of the invention on LPS-induced TNFα shedding in vitro.

In the following study, an ELISA-based TNFα release assay was performed to verify the inhibitory effect of the purified antibody 4H8-E3 of the invention on LPS-induced release of endogenous TNFα from human THP-1 macrophage cells.

Briefly, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were treated with 4 μg/ml TBS with a mouse anti-human TNFα capture antibody (as part of the DuoSet ELISA kit). provided) overnight. On day 2, the capture antibody solution was removed and the MaxiSorp® plates were blocked with 1% BSA in 300 μl TBS per well for 3 hours at room temperature. Meanwhile, 20,000 THP-1 (American Type Culture Collection, USA) cells in 80 μl of normal growth medium were added to each well of a Greiner CELLSTAR V-bottom 96-well plate (Thermo Fisher Scientific, USA). Standard growth medium (sample volume at 10 μM final concentration) supplemented with 50 μM batimastat (BB94, Abcam, UK) as positive control, 50 μg mouse IgM antibody (clone PFR-03, Sigma-Aldrich, USA) as isotype control /ml (100 μl sample volume at a final concentration of 10 μg/ml) or purified antibody 4H8-E3 of the invention at 50 μg/ml (100 μl sample volume at a final concentration of 10 μg/ml) at 37 ^° C., 5% CO ₂ for 30 minutes. incubated.

For stimulation controls, 20 μl of standard growth medium without test substance was added. Cells (except unstimulated controls) were then plated with LPS (Sigma-Aldrich, USA) in 300 ng/ml growth medium at 20 μl per well to a final concentration of 50 ng/ml at 37° C., 5% CO ₂ . Stimulated for 2 hours. The 96-well plate was then centrifuged to pellet the cells. In parallel, the blocking buffer was removed from the MaxiSorp® plates and the plates were washed 4 times with 350 μl per well of TBS-T (Karl Roth, Germany) on a 96-head plate washer (Tecan Group, Switzerland). washed twice. To avoid drying, 30 μl of TBS was immediately added to each well of the MaxiSorp® plate before transferring 70 μl of cell-free supernatant per sample. In addition, 100 μl of recombinant human TNFα protein (provided as part of the DuoSet ELISA kit) diluted in TBS to defined concentrations was added to the plate as a standard reference.

100 μl per well of biotinylated goat anti-human TNFα detection antibody (provided as part of the DuoSet ELISA kit) in 50 ng/ml TBS was then added, protected from direct light, and the plates were incubated for 2 hours at room temperature. After 4 washes with 350 μl per well of TBS-T (Carl Roth, Germany) on a 96-head plate washer (Tecan Group, Carl Roth, Switzerland), carefully removing all traces of buffer from the 4th cycle onwards. , 100 μl of streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS was added to each well, again protected from direct light, and the plate was incubated at room temperature for 30 minutes. Wash again 4 times with 350 μl per well of TBS-T (Carl Roth, Germany) on a 96-head plate washer (Tecan Group, Switzerland), carefully removing all traces of buffer from the 4th cycle onwards, then place at room temperature. 100 μl of AttoPhos substrate solution (Promega, USA) was added for 1 hour incubation in the dark. Fluorescence of each well was collected with an excitation wavelength of 435 nm and an emission wavelength of 555 nm using an infinite M1000 PRO (Tecan Group, Switzerland) microplate reader.

Figure 6 shows representative results of this experiment showing the effect of test article on LPS-induced TNFα release from THP-1 cells in absolute numbers (Figure 6A) and percent inhibition (Figure 6B). Batimastat (BB94), as a small molecule inhibitor of metalloproteases, served as a positive control, with 92.5% inhibition of LPS triggering the release of TNFα, whereas the presence of an IgM isotype control had a significant effect on TNFα shedding. do not affect In contrast, an equivalent concentration of the purified antibody 4H8-E3 of the invention inhibits LPS-induced TNFα release from THP-1 cells by 62.6%.

Example 12: Determination of the IC50 of the antibody 4H8-E3 of the invention against LPS-induced TNFα shedding in vitro

Extending the functional analysis, an ELISA-based TNFα release assay was performed to determine the half-maximal inhibitory concentration (IC50) of the purified antibody 4H8-E3 of the invention on LPS-induced release of endogenous TNFα from human THP-1 macrophage cells. It was determined.

Briefly, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were treated with a mouse anti-human TNFα capture antibody (as part of the DuoSet ELISA kit) at 4 μg/ml TBS. provided) was coated at 100 μl per well overnight at 4°C. On day two, the capture antibody solution was removed and the MaxiSorp® plates were blocked with 300 μl 1% BSA in TBS per well for 3 hours at room temperature. On the other hand, 20,000 THP-1 (USA Type Culture Collection) cells were seeded into each well of a Greiner CELLSTAR V-bottom 96-well plate (Thermo Fisher Scientific, USA) in 80 μl of normal growth medium, and purified according to the present invention. Antibody 4H8-E3, 400.00 μg/ml, 307.69 μg/ml, 236.68 μg/ml, 182.06 μg/ml, 140.05 μg/ml, 107.73 μg/ml, 82.87 μg/ml, 63.74 μg /ml, 49.03 μg/ml, 37.71 μg/ml, 29.01 μg/ml, 22.31 μg/ml, 17.16 μg/ml, 13.20 μg/ml, 10.15 μg/ml, 7.81 μg/ml , 6.01 μg/ml, 4.62 μg/ml, 3.55 μg/ml, 2.73 μg/ml, 2.10 μg/ml, 1.61 μg/ml, 1.24 μg/ml, 0.95 μg/ml, 0 .73 μg/ml, 0.56 μg/ml, and 0.43 μg/ml (final concentrations of about 80.00 μg/ml, 61.53 μg/ml, 47.33 μg/ml, 36.41 μg/ml, respectively for a sample volume of 100 μl). 28.01 μg/ml, 21.54 μg/ml, 16.57 μg/ml, 12.74 μg/ml, 9.80 μg/ml, 7.54 μg/ml, 5.80 μg/ml, 4.46 μg/ml, 3. 43 μg/ml, 2.64 μg/ml, 2.03 μg/ml, 1.56 μg/ml, 1.20 μg/ml, 0.92 μg/ml, 0.71 μg/ml, 0.54 μg/ml, 0.42 μg/ml standard growth medium supplemented with 20 μl per well of was preincubated for 30 minutes at 37°C, 5% _CO2 .

Cells (except unstimulated controls) were then plated with LPS (Sigma-Aldrich, USA) in 300 ng/ml growth medium at 20 μl per well to a final concentration of 50 ng/ml at 37° C., 5% CO _{2 .} Stimulated for 3 hours. The 96-well plate was then centrifuged to pellet the cells. In parallel, the blocking buffer was removed from the MaxiSorp® plates and the plates were washed 4 times with 350 μl per well of TBS-T (Karl Roth, Germany) on a 96-head plate washer (Tecan Group, Switzerland). washed twice. To avoid drying, 30 μl of TBS was immediately added to each well of the MaxiSorp® plate before transferring 70 μl of cell-free supernatant per sample. In addition, 100 μl of recombinant human TNFα protein (provided as part of the DuoSet ELISA kit) diluted in TBS to defined concentrations was added to the plate as a standard reference. 100 μl per well of biotinylated goat anti-human TNFα detection antibody (provided as part of the DuoSet ELISA kit) in 50 ng/ml TBS was then added, protected from direct light, and the plates were incubated for 2 hours at room temperature.

After 4 washes with 350 μl per well of TBS-T (Carl Roth, Germany) on a 96-head plate washer (Tecan Group, Carl Roth, Switzerland), carefully removing all traces of buffer from the 4th cycle onwards. , 100 μl of streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS was added to each well, again protected from direct light, and the plate was incubated at room temperature for 30 minutes. Wash again 4 times with 350 μl per well of TBS-T (Carl Roth, Germany) on a 96-head plate washer (Tecan Group, Switzerland), carefully removing all traces of buffer from the 4th cycle onwards, then place at room temperature. 100 μl of AttoPhos substrate solution (Promega, USA) was added for 1 hour incubation in the dark. Fluorescence of each well was collected with an excitation wavelength of 435 nm and an emission wavelength of 555 nm using an infinite M1000 PRO (Tecan Group, Switzerland) microplate reader.

Figure 7 shows representative results of this experiment. The titer of the purified antibody 4H8-E3 of the invention results in concentration-dependent inhibition of TNFα release from THP-1 cells. Applying Prism8 software (GraphPad software, USA), the respective IC50 value for the antibody 4H8-E3 of the invention is calculated to be 6.48 nM.

Literature

arrangement

The sequences below form part of the disclosure of the present application. A WIPOS ST 25 compatible electronic sequence listing is also provided with this application. For the avoidance of doubt, in the event of any discrepancy between the sequences in the table below and those in the electronic sequence listing, the sequences in this table shall be deemed correct.

Claims (29)

A protein binder that binds to human iRhom2, wherein binding to human iRhom2 inhibits and/or reduces TACE/ADAM17 activity. 2. A protein binder according to claim 1, which is a monoclonal antibody, or a target-binding fragment or derivative thereof which retains target-binding ability, or an antibody mimetic. 3. The protein binder of claim 1 or 2, wherein inhibition or reduction of TACE/ADAM17 activity is caused by interference with iRhom2-mediated TACE/ADAM17 activation. 4. A protein binder according to any one of the preceding claims, wherein the antibody inhibits or reduces TNF[alpha] shedding. Human iRhom2 to which the protein binder binds
a) comprising the amino acid sequence set forth in SEQ ID NO: 16, or b) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 16, provided that it maintains iRhom2 activity,
A protein binder according to any one of the preceding claims. 4. A protein binder according to any one of the preceding claims, which binds to the juxtamembrane domain adjacent to transmembrane domain 1 (TMDl) of human iRhom2. 3. Any of the preceding claims which binds to an amino acid sequence of human iRhom2 comprising a) comprising at least the amino acid sequence set forth in SEQ ID NO:3, or b) comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO:3. or the protein binder according to claim 1. 3. A protein binder according to any one of the preceding claims which binds to one or more amino acid sequences of human iRhom2 each comprising one or more amino acids within the amino acid sequence set forth in SEQ ID NO:3. A431, Q432, H433, V434, T435, T436, Q437, L438, V439, L440, R441, N442, K443, G444, V445, Y446, E447, S448, V449, K450, Y451, I452, Q453, Q454, E 455, N456, F457, W458, V459, wherein said amino acid residue numbering refers to the amino acid sequence set forth in SEQ ID NO: 16 (human iRhom2). 10. Protein binder according to any one of the clauses. 4. A protein binder according to any one of the preceding claims, which is not cross-reactive with human iRhoml or the juxtamembrane domain adjacent to transmembrane domain 1 (TMDl). 4. A protein binder according to any one of the preceding claims, which is an antibody in at least one format selected from the group consisting of IgG, scFv, Fab, (Fab)2. 4. A protein binder according to any one of the preceding claims, which is an antibody with an isotype selected from the group consisting of IgG, IgM. 4. A protein binder according to any one of the preceding claims, which is a murine, chimerized, humanized or human antibody. A protein binder according to any one of the preceding claims, which is antibody 4H8-E3. 4. A protein binder according to any one of the preceding claims, which is an antibody comprising the CDRs of variable domains or 4H8-E3. A protein binder according to any one of the preceding claims, wherein the protein is
a) comprises a pair of heavy/light chain complementarity determining regions (CDRs) contained in the heavy/light chain variable region sequence pairs set forth in SEQ ID NOS: 33 and 40;
b) the following sequences HC CDR1 (SEQ ID NO: 34 or 37), HC CDR2 (SEQ ID NO: 35 or 38), HC CDR3 (SEQ ID NO: 36 or 39), LC CDR1 (SEQ ID NO: 41 or 44), LC CDR2 (SEQ ID NO: 42 or 45), and a set of heavy/light chain complementarity determining regions (CDRs) including LC CDR3 (SEQ ID NO: 43 or 46);
c) comprising the heavy/light chain complementarity determining regions (CDRs) of b), wherein at least one of the CDRs has up to 3 amino acid substitutions relative to each of SEQ ID NOS: 34-39 or 41-46; and /or d) comprises the heavy/light chain complementarity determining regions (CDRs) of b) or c), provided that at least one of the CDRs is 66% or greater relative to SEQ ID NOS: 34-39 or 41-46, respectively having sequence identity,
A protein binder, wherein said CDRs are embedded in a suitable protein framework such that they can bind human iRhom2 with sufficient binding affinity and inhibit or reduce TACE/ADAM17 activity. 4. A protein binder according to any one of the preceding claims, wherein the framework is a human VH/VL framework. A protein binder according to any one of the preceding claims comprising:
a) heavy/light chain variable domain (VD), HC VD (SEQ ID NO:33) and LC VD (SEQ ID NO:40),
b) the heavy/light chain variable domain (VD) of a), provided that the HC VD has ≧80% sequence identity to the respective SEQ ID NO: 33 and/or the LC VD has the respective ≧80% sequence identity to SEQ ID NO: 40, or c) have the heavy/light chain variable domains (VD) of a) or b), provided that at least one of the HC VD or LC VD is , with up to 10 amino acid substitutions relative to each of SEQ ID NOs: 33 and/or 40;
Said protein binder binds human iRhom2 with sufficient binding affinity and is still capable of inhibiting or reducing TACE/ADAM17 activity. 4. A protein binder according to any one of the preceding claims, wherein at least one amino acid substitution is a conservative amino acid substitution. The protein binder has a target binding affinity for iRhom2 of ≧50% as measured by SPR compared to that of the protein binder according to any one of the preceding claims and/or any one of the preceding claims 5. A protein binder according to any one of the preceding claims, having at least one of ≧50% of an inhibitory or reducing effect on TACE/ADAM17 activity of the protein binder according to any one of the preceding claims. A protein binder that competes for binding to iRhom2 with a protein binder according to any one of the preceding claims. A protein binder that binds to essentially the same or the same epitope on iRhom2 as the protein binder of any one of the preceding claims. A protein binder according to any one of claims 11 to 22, which is a monoclonal antibody, or a target-binding fragment or derivative thereof which retains target-binding ability, or an antibody mimetic. A nucleic acid molecule encoding a binding agent according to any one of the preceding claims. For the treatment of a human or animal subject diagnosed with, suffering from, or at risk of developing an inflammatory condition, or for the prevention of such a condition. Use of the protein binder (for the manufacture of a medicament) according to any one of 1-23. A pharmaceutical composition comprising a protein binder according to any one of claims 1-23 and optionally one or more pharmaceutically acceptable excipients. A combination comprising a protein binder according to any one of claims 1 to 23 or a pharmaceutical composition according to claim 26 and (ii) one or more therapeutically active compounds. A method of treating or preventing an inflammatory condition comprising (i) a protein binder according to any one of claims 1-23, (ii) a pharmaceutical composition according to claim 26 or (iii) claim 27. A method comprising administering to a human or animal subject a therapeutically sufficient amount of the combination described in . a) a composition according to any one of claims 1 to 23, a pharmaceutical composition according to claim 26 or a combination according to claim 27, b) a composition, composition or combination for administering A treatment kit of parts containing the device and c) instructions for use.